A. G. R. Sheil

Evidence that apoptosis of activated T cells occurs in spontaneous tolerance of liver allografts and is blocked by manipulations which break tolerance

BACKGROUND Fully allogeneic liver grafts from piebald virol glaxo to dark agouti rats are spontaneously tolerated, whereas kidney transplants between these strains are rejected. Liver tolerance is broken by donor irradiation or peritransplant corticosteroid treatment of recipient rats, both of which interfere with the activation of recipient cells. METHODS In this study we used a combination of immunohistochemical staining, reverse transcription-polymerase chain reaction, and terminal deoxynucleotide transferase-mediated dUTP nick end labeling and Annexin-V apoptosis assays to compare donor cell migration, cytokine profiles, and leukocyte apoptosis in grafts and lymphoid organs from tolerant liver and rejecting kidney recipients. We then examined the effect on apoptosis of treatments which abrogate liver tolerance. RESULTS Liver transplantation in this tolerant strain combination is accompanied by rapid migration of many passenger leukocytes to the recipient spleen and lymph node, concurrent with a marked but transient increase in the amount of mRNA for the cytokines interleukin-2 and interferon-gamma. Apoptotic cells appear promptly in the spleen, their numbers reaching a peak 2 days earlier than has been previously shown for the graft infiltrate. Both CD4+ and CD8+ T cells undergo apoptosis and apoptotic cells are most concentrated among CD25+ T cells. In contrast, renal transplant rejection is associated with limited donor cell migration to lymphoid tissues and significantly less up-regulation of interleukin-2 and interferon-gamma in the spleen. Few apoptotic cells are detected in spleen or graft infiltrate during rejection, whereas apoptotic renal tubular and glomerular cells are found from day 5. Either recipient steroid treatment or donor irradiation significantly reduced the number of apoptotic cells in liver graft infiltrates and recipient spleen. CONCLUSIONS Taken together, these findings suggest that a mechanism akin to activation-induced cell death, with apoptosis of alloreactive recipient cells may be responsible for the induction of spontaneous liver transplant tolerance.

Kinetics of intragraft cytokine expression, cellular infiltration, and cell death in rejection of renal allografts compared with acceptance of liver allografts in a rat model : Early activation and apoptosis is associated with liver graft acceptance

BACKGROUND Liver transplants in the rat strain combination PVG-to-Dark Agouti are spontaneously tolerated, whereas kidney transplants in the same strain combination are rejected in 7-9 days. METHODS To identify organ-specific differences that might yield further information about the mechanism of tolerance induction in this strain combination, liver or kidney grafts, spleen, and draining lymph nodes were harvested at days 1, 3, 5, and 7, and examined by immunohistochemistry, terminal deoxynucleotide transferase-mediated dUTP nick end labeling assay, and reverse transcriptase-polymerase chain reaction for interferon-gamma, interleukin (IL)-2, IL-4, and IL-10. RESULTS Renal allograft rejection was associated with the progressive development of an intense mononuclear cell infiltrate. Markers of lymphocyte activation and cytokine up-regulation appeared from day 3, and many apoptotic parenchymal cells were noted on days 5-7, at the peak of rejection. Conversely, liver allograft tolerance was associated with more rapid infiltration by activated T cells and earlier increases in cytokine expression, but with a more limited degree of cellular infiltration. Concurrent with the early activation, high levels of apoptosis were found in areas of leukocyte infiltrate, paralleling the disappearance of activated T cells from the graft between days 3 and 5. CONCLUSIONS Apoptosis of infiltrating leukocytes in liver allografts may represent an important process in the induction of spontaneous liver transplant tolerance and may underlie the abortive nature of the effector response observed within tolerated livers. In contrast, activated cells in renal allografts in the same strain combination survive and proliferate, express high levels of cytokines, and are efficient in bringing about graft destruction.

A short course of methylprednisolone immunosuppression inhibits both rejection and spontaneous acceptance of rat liver allografts.

BACKGROUND The effects of immunosuppressive drugs on transplant tolerance have not been extensively studied, although their effect on rejection is well established. METHODS We examined the effects of a short course of treatment with the immunosuppressive drug methylprednisolone (MP) on the survival of PVG liver allografts in Dark Agouti (DA) recipients that accepted the livers and in Lewis recipients that rejected the livers. Infiltration of liver allografts was examined by immunohistochemical staining of liver sections, and apoptosis was measured by terminal deoxynucleotide transferase-mediated dUTP nick end labeling. RESULTS A 5-day course of MP (days 0 to 4) led to rejection of four of six livers (mean survival time [MST] 99 days) in DA recipients compared with long-term survival (MST >100 days) in untreated animals. Delayed administration of MP (days 3 to 7) exacerbated rejection in DA recipients, and all eight animals rejected the graft (MST 68.5 days). Treatment of Lewis recipients with MP did not significantly prolong survival when administered from days 0 to 4 (MST 13 days), although delay of administration improved the outcome. Treatment from days 3 to 7 resulted in an MST of 21 days, whereas treatment from days 7 to 11 resulted in an MST of 41.5 days. MP treatment from day 3 to day 7 reduced T cells and interleukin 2 receptor-expressing cells but increased the numbers of apoptotic cells infiltrating both DA and Lewis strain allografts. CONCLUSIONS These results show that immunosuppression with MP inhibits both spontaneous tolerance and rejection of liver allografts in a rat model and question the efficacy of administering MP to all liver allograft recipients from the time of transplantation.

A Large, Single Center Investigation Of The Immunogenetic Factors Affecting Liver Transplantation

BACKGROUND Reports on the relevance of immunogenetic factors in liver transplantation are often conflicting or inconclusive. We have, therefore, investigated a range of factors that may underlie liver graft survival. METHODS The influences of HLA, flow cytometric, and enhanced cytotoxic crossmatching and immunoglobulin (Ig)A levels on graft survival, and acute and chronic rejection were investigated for a single center involving 446 patients over 13 years. RESULTS The effect of HLA mismatching on graft survival was significant (P<10(-2)) and was reversed in recipients with autoimmune diseases (P<0.5x10(-2)), whereas the effect of HLA mismatches on the level of acute rejection was detrimental in all recipients. There was a significant effect of a positive cytotoxic crossmatch on 3-month (P<10(-5)) and 1-year (P<10(-4)) graft survival, and an additional effect of the flow cytometric crossmatch was seen for chronic rejection (P<10(-2)) and acute rejection (P<10(-2)). Recipients with HLA-A1,B8,DRB1*0301 had higher levels of acute rejection (P<0.5x10(-2)), and recipients who received an ABO compatible-nonidentical transplant have a significantly higher risk (P<10(-2)) of developing chronic rejection. Finally, the beneficial effect of high serum IgA and, specifically, IgA anti Fab, seen in renal transplants was not evident in liver transplants, and in fact the opposite may be true, at least for acute rejection (P<0.5x10(-2)). CONCLUSIONS By separating the recipients with autoimmune disease from other patients and by including acute and chronic rejection as outcome parameters, we have used the power of a large single-centre study to delineate the significance of some of the important immunogenetic factors involved in liver transplantation.

Biliary strictures after liver transplantation: Clinical picture, correlates and outcomes

We retrospectively examined 154 adults to ascertain the frequency, site of and pre‐disposing factors for biliary strictures after liver transplantation, as well as their management and clinical outcome. Twenty patients (12.5%) were identified with biliary strictures; 16 were non‐anastomotic and four were anastomotic strictures. The median time from transplantation to stricture diagnosis was 17 weeks (range 3–366). Of the 16 non‐anastomotic strictures, six were intrahepatic, eight hilar and two extrahepatic (donor bile duct). A control group (n = 32) of patients transplanted immediately before and after index cases was used to examine for correlates in patients with non‐anastomotic strictures. At the time of diagnosis in the non‐anastomotic index cases, there was a higher incidence of: (i) biliary sludge (63 vs 0%; P< 0.001); and (ii) clinical cholangitis (75 vs 0%; P< 0.001) compared with controls. Primary sclerosing cholangitis was more often the diagnosis in index patients with non‐anastomotic strictures compared with controls (31 vs 9%; P<0.05). There were no differences between index patients and controls (non‐anastomotic group) in ABO blood group non‐identity, cold allograft ischaemia time, use of OKT3 (murine monoclonal antibody to CD3) and hepatic artery thrombosis. Of 15 patients treated with balloon dilatation, seven required stent insertion although none have required surgery. As determined by liver function tests, there was evidence of persisting graft dysfunction in index patients compared with controls (SAP 381 vs 112 U/L, P< 0.001; GGT 529 vs 80 U/L, P< 0.001), but there was no difference in survival during a median follow‐up time of 16 months (range: 3–48 months) from stricture diagnosis. In conclusion, biliary strictures tend to occur within 6 months of transplantation and are an important cause of ongoing graft dysfunction. Non‐anastomotic strictures were more common in patients requiring transplantation for primary sclerosing cholangitis.

RENAL AUTOTRANSPLANTATION FOR SEVERE LOIN-PAIN/HAEMATURIA SYNDROME

Most forms of therapy for the loin-pain/haematuria syndrome are unsuccessful, though nerve-block procedures and renal denervation sometimes provide temporary relief. Three young patients, with pain so severe that they were dependent on narcotics, were treated for this syndrome. All three had been repeatedly admitted to hospital. Loin pain was unilateral in two patients and predominantly so in the third. Renal function, excretion urography, and angiography were normal, but renal biopsy specimens showed deposition of the third component of complement in the renal arterioles. All were treated with renal autotransplantation; the kidney causing pain (or the one causing the most pain) was completely excised and reimplanted in the iliac fossa. Two patients had complete relief of pain and the third almost complete relief, despite recurrence of haematuria in all three. It is too early to determine whether this major procedure is justified in the treatment of the loin-pain/haematuria syndrome, but the early results are encouraging.

Conversion of pancreas allograft rejection to acceptance by liver transplantation

BACKGROUND Liver allografts of PVG(RT1c)-->DA(RT1a) are spontaneously accepted, whereas pancreas, heart, and kidney allografts are rejected. Our previous studies have shown that simultaneous liver and pancreas transplantation prevents pancreas allograft rejection. The aim of this study was to examine the effect of liver transplantation on subsequent pancreas allografts and on ongoing pancreas rejection. METHODS Heterotopic, duct-ligated segmental pancreas grafts were transplanted into streptozotocin-induced diabetic recipients (60 mg/kg body weight i.p.) with or without orthotopic liver grafting at different times. Experimental design was as follows. Group 1 received PVG-->PVG pancreas syngrafts (n=6); group 2, PVG-->DA pancreas allografts (n=7); groups 3-5, PVG-->DA pancreas allografts followed by liver transplantation on day 2 (n=6), on day 4 (n=5), and on day 6 (n=5), respectively, group 6, PVG-->DA pancreas allografts after liver transplantation at 4 weeks (n=6). RESULTS The results showed that pancreas allografts in group 2 were rejected from postoperative day 7 to 13. Liver transplantation prevented subsequent pancreas allograft rejection in group 6. Ongoing pancreas rejection was reversed by liver transplantation with subsequent graft acceptance in groups 3-5. Significant graft-infiltrating lymphocyte apoptosis was demonstrated at 2 weeks in pancreas transplants associated with liver grafting. Graft-versus-host disease was not detected in the pancreas recipients. CONCLUSIONS We conclude that pancreas allografts in the PVG-->DA combination are rejected rapidly with median survival time of 9 days. Liver transplantation can protect subsequent pancreas grafts from rejection and reverse ongoing pancreas graft rejection with subsequent pancreatic acceptance. Graft-infiltrating lymphocyte apoptosis may be associated with the process of graft acceptance.

Case Report: Delayed resolution of severe pulmonary hypertension after isolated liver transplantation in a patient with cirrhosis

Pulmonary hypertension is now recognized to be a rare association of liver disease and portal hypertension. This report describes the slow resolution of symptomatic pulmonary hypertension in a 33‐year‐old woman with cirrhosis who underwent isolated liver transplantation. The patient survived the surgery and perioperative period without significant haemodynamic compromise. After liver transplantation, the patient was monitored with regular Doppler echocardiography. By 27 months the pulmonary hypertension had almost completely resolved. This observation is important, as it suggests that patients with severe pulmonary hypertension who survive the perioperative period may have an excellent outcome, although resolution may be slow.

CONTROLLED CLINICAL TRIAL OF ANTILYMPHOCYTE GLOBULIN IN PATIENTS WITH RENAL ALLOGRAFTS FROM CADAVER DONORS

Abstract A controlled clinical trial of antilymphocyte globulin (A.L.G.) for adjuvant immunosuppression in recipients of renal allografts from cadaver donors is described. A.L.G. of horse and goat origin was used. As far as possible patients were chosen so that influences known to affect the outcome of renal transplantation were similar in the A.L.G.-treated and control groups. The trial also met the requirements of the use of A.L.G. with proven immunosuppressive properties, concurrent observation of treated and control patients, and uniform treatment of all recipients with standard immunosuppressive drugs. There were fifty-four A.L.G.-treated patients and forty-six controls. Graft function continues in forty-five (83%) A.L.G.-treated patients and thirty (65%) controls. All survivors are between 4 and 26 months after operation; mean survival in both groups is 13 months. When grafts with seriously impaired function and those which ceased function are considered as failures, ten (19%) grafts in the A.L.G. group failed compared with nineteen (41%) controls. Five (9%) grafts failed because of rejection in the treated group and fourteen (30%) in the control. Prolonged good function was achieved in thirty-nine (72%) of A.L.G.-treated patients and twenty-four (52%) controls. There was less mortality from sepsis in the A.L.G.-treated group, no malignant tumour has developed in either group. The complications of therapy were not serious, and, although horse globulin was dose-limited because of side-effects, goat globulin was nontoxic and without apparent dose limitations. It is concluded that A.L.G. preparations can be safe and immunosuppressive, that such globulin significantly impairs human rejection processes, and that this is manifested as decreased rejection of grafts and increased proportions of grafts which achieve good function. It is suggested that improved immunosuppression from A.L.G. therapy also results in delayed chronic rejection, lower mortality from infective complications, and increased safety with grafts with poor early postoperative function because of ischaemic damage. A.L.G. should be used to treat recipients of renal allografts from cadaver donors.

PROGNOSTIC VALUE OF INTRAOPERATIVE BLOOD-FLOW MEASUREMENTS IN FEMOROPOPLITEAL BYPASS VEIN-GRAFTS

Abstract A prospective study has been carried out on 40 patients having saphenous-vein bypass grafts for femoropopliteal arterial obstruction. Records were kept of intraoperative mean blood-flow, measured with a square-wave electromagnetic flow-meter. Early graft-occlusion was defined as occurring within the first three months. A highly significant difference was found between the mean flow of grafts which occluded early and that of those that remained patent: an intraoperative graft-flow of less than 60 ml. per minute carried an 80% chance of early thrombosis, while a flow of 60 ml. per minute or more carried an 80% chance of patency for three months or more. The observed probability of early graft-occlusion fell as the intraoperative flow rose. The rank score based on the intraoperative mean flow correlated significantly with the ranked observed probability of early graft-occlusion.